This project is designed to investigate the molecular factors that determine the mechanism of steroid hormone binding to specific macromolecular binding sites. The steroids which will be synthesized to conduct these studies will ultimately be tested for their affects on the female reproductive tract in the expectation of illuminating the connection between the mechanism of steroid binding to macromolecules and the mechanisms of steroid hormone action. Ovine and bovine 20alpha-hydroxysteroid dehydrogenase, from fetal erythrocytes, will be isolated by affinity chromatography and crystallized by electrophoretic diffusion. The enzyme active site amino acid composition and topography will be characterized by affinity labeling experiments using isomeric (2'-3H) bromoacetoxyprogesterone derivatives previously synthesized by us, and also with a newly proposed series of bifunctionally reactive steroid analogs. We shall examine the substrate activity of protein conjugated progesterone derivatives toward pure 20alpha-hydroxysteroid dehydrogenase to further examine the nature of specific protein-steroid-protein interactions. New progesterone-protein conjugates will be synthesized, which will be stable under in vivo mammalian physiological conditions, and these substances will be used to determine whether steroids bound to proteins can be directly metabolized. We will also determine whether steroid-protein conjugates, which are not likely to undergo intranuclear transport, can evoke a hormone response in target tissue.